The intrinsically disordered protein alpha-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD). We show here that the native state of alpha-synuclein consists of a broad distribution of conformers with an ensemble-averaged hydrodynamic radius significantly smaller than that expected for a random coil structure. This partial condensation is driven by interactions between the highly charged C-terminus and a large hydrophobic central region of the protein sequence. We suggest that this structure could inhibit the formation of alpha-synuclein aggregates, which are thought to be the cytotoxic species responsible for neurodegeneration in PD.

Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells. We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (>400 g͞liter) of glucose, BSA, or ovalbumin. Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM. The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions. M ost proteins require a defined three-dimensional structure to perform their function. Intrinsically disordered proteins seem paradoxical because they lack stable structure, yet they play key roles in diverse biological processes including signal transduction, transcription, and neurodegenerative diseases (1, 2). Here, we report results from studies of the so-called intrinsically disordered protein, FlgM, a 97-residue polypeptide from Salmonella typhimurium that regulates flagellar synthesis by binding the transcription factor , as shown by the disappearance of crosspeaks from residues in the C-terminal half of FlgM in the FlgM-28 complex (8). This bipartite behavior (i.e., disappearance of crosspeaks from the C-terminal half with retention of crosspeaks from the Nterminal half) provides a valuable built-in control for studying the response of FlgM to different solution conditions. We discuss our results in terms of two types of intrinsically disordered proteins: those that gain structure under crowded conditions and those that do not. FlgM is an example of both types in a single protein. Materials and MethodsFlgM was overexpressed and purified as described (4,8). In vitro NMR data were acquired by using a 0.4 mM uniformly 15 Nenriched sample in 10 mM sodium acetate, pH 5.0͞10 mM sodium chloride͞0.02% sodium azide͞10.0% (vol͞vol) D 2 O at 25°C. For the crowding experiments, glucose, BSA, or ovalbumin were incorporated into the sample. The sample used for live-cell NMR spectroscopy was obtained by overexpressing 15 N-labeled FlgM in BL21 Gold Escherichia coli bacteria and by preparing as described (9). Two-dimensional gradient-enhanced ResultsThe HSQC spectrum of 15 N-enriched FlgM in dilute solution is contrasted with the spectrum in living E. coli cells in Fig. 1. In-cell NMR experiments are possible because FlgM is overexpressed upon induction (Ϸ100 mg of FlgM can be purified from 1 liter of saturated culture). The overexpression allows the FlgM spectrum to be observed on top of signals arising from other 15 N-enriched proteins in the cell, which contribute a uniform background (9). As shown in Fig. 1, about half These data suggest that the C-terminal portion of FlgM is structured in cells, but the N-terminal portion remains unstructured. We also performed important controls. ...

Parkinson's disease (PD) is a neurodegenerative disorder affecting an estimated 4 million people worldwide. Intracellular proteinaceous inclusions called Lewy bodies are the histological hallmarks of PD and are primarily composed of aggregated ␣-synuclein (␣Syn). Although the detailed mechanisms remain unclear, mounting evidence suggests that the misfolding of ␣Syn into prefibrillar and fibrillar species is the driving force responsible for cellular toxicity. We show here that the molecular chaperone heat shock protein (Hsp) 70 strongly inhibits ␣Syn fibril formation via preferential binding to prefibrillar species. Moreover, our studies reveal that Hsp70 alters the characteristics of toxic ␣Syn aggregates and indicate that cellular toxicity arises from the prefibrillar forms of ␣Syn. This work therefore elucidates a specific role of Hsp70 in the pathogenesis of PD and supports the general concept that chaperone action is a crucial aspect in protecting against the otherwise damaging consequences of protein misfolding.

a-and b-synuclein are closely related proteins, the first of which is associated with deposits formed in neurodegenerative conditions such as Parkinson's disease while the second appears to have no relationship to any such disorders. The aggregation behavior of a-and b-synuclein as well as a series of chimeric variants were compared by exploring the structural transitions that occur in the presence of a widely used lipid mimetic, sodium dodecyl sulfate (SDS). We found that the aggregation rates of all these protein variants are significantly enhanced by low concentrations of SDS. In particular, we inserted the 11-residue sequence of mainly hydrophobic residues from the non-amyloid-b-component (NAC) region of a-synuclein into b-synuclein and show that the fibril formation rate of this chimeric protein is only weakly altered from that of b-synuclein. These intrinsic propensities to aggregate are rationalized to a very high degree of accuracy by analysis of the sequences in terms of their associated physicochemical properties. The results begin to reveal that the differences in behavior are primarily associated with a delicate balance between the positions of a range of charged and hydrophobic residues rather than the commonly assumed presence or absence of the highly aggregation-prone region of the NAC region of a-synuclein. This conclusion provides new insights into the role of a-synuclein in disease and into the factors that regulate the balance between solubility and aggregation of a natively unfolded protein.